Opiates are widely used as analgesic agents, but one of their major side- effects in addiction. Opiates induce their biological effects by interacting with three major subtypes or receptor, the mu, delta, and kappa. The neuropeptide, enkephalins and dynorphins interact specifically with delta and kappa receptors, respectively. We have recently cloned the mouse kappa and delta opiate receptor cDNA. We propose to use these cDNA to elucidate the molecular mechanisms of action of the endogenous neurotransmitters and of the clinically used pharmacological agents that act on these receptors to induce their biological effects. The amino acid sequences of the two opiate receptors are 60% identical with major differences in the N-terminal regions. We will use site-directed mutagenesis to test the hypothesis based mutagenesis to identify the domains of the kappa and delta receptors linked to G proteins, which couple these receptors to adenylyl cyclase, Ca++ and K+ channels. To determine which G proteins associate with the cloned opiate receptors, we will employ an immunoprecipitation technique using selective antisera against the subtypes of Gi and Go to detect specific opiate receptor/G protein complexes from cell lines in which we have stably expressed the delta and kappa receptors. We will also use these cell lines to determine whether each cloned opiate receptor couples to Ca++ and K+ channels employing patch-clamp electrophysiological techniques. These studies will identify functional domains of the opiate receptors and the cellular effector systems linked to each receptor. Chronic treatment of animals with delta or kappa selective agonists induces tolerance. The molecular basis of tolerance has been associated with opiate receptor desensitization. To study delta and kappa agonist induced tolerance at a cellular level, we will identify the biochemical events involved in delta and kappa receptor desensitization. In initial studies, we have shown that the cloned kappa and delta receptors desensitize following agonist treatment and that the enzyme beta- adrenergic receptor kinase (BARK) is involved in the desensitization. We will determine whether this kinase catalyzes the phosphorylation of the opiate receptors to cause desensitization and will investigate whether BARK is also involved in delta and kappa receptor down- regulation. To test whether a neuroadaptive response to chronic opiate use is changes in opiate receptor gene expression, we will determine whether prolonged treatment of cells in culture or rodents with subtype selective opiate agonists alters delta or kappa receptor mRNA levels using Northern analysis and in situ hybridization histochemistry. These studies will determine the molecular basis of kappa and delta receptor desensitization and tolerance and will investigate the long-term cellular consequences of chronic opiate use. The overall goal of this proposal is to identify the cellular mechanisms by which kappa and delta opiate induce there biological and clinical effects and will reveal the molecular basis for the side-effects of chronic use of agonists at the receptors.